This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of these polynucleotides and polypeptides, processes for making these polynucleotides and these polypeptides, and their variants and derivatives and antagonists of the polypeptides; and uses of these polynucleotides, polypeptides, variants, derivatives and antagonists, in particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of spsA, hereinafter referred to as xe2x80x9cspsAxe2x80x9d.
The majority of proteins that are translocated across one or more membranes from the site of synthesis are initially synthesized with an N-terminal extension known as a signal, or leader, peptide (Wickner, W., et al, (1991). Ann. Rev. Biochem. 60:101-124). Proteolytic cleavage of the signal sequence to yield the mature protein occurs during, or shortly after, the translocation event and is catalyzed in both prokaryotes and eukaryotes by enzymes known as signal, or leader, peptidases (herein xe2x80x9cSPasesxe2x80x9d). The bacterial SPases are membrane proteins consisting of a single polypeptide anchored to the membrane by one (Gram-positive (herein xe2x80x9cG+xe2x80x9d) and Gram-negative (herein Gxe2x88x92) bacteria) or two (Gxe2x88x92bacteria) transmembrane sections. Predicted amino acid sequences of bacterial SPases show a high level of similarity and are known for Escherichia coli (Wolfe, P. B, at al, (1983) J. Biol. Chem. 258:12073-12080),Pseudomonas fluorescens (Black, M. T., et al, (1992). Biochem. J. 282:539-543), Salmonella typhimurium (van Dijl, J. M., et al, (1990). Mol. Gen. Genet. 223:233-240), Haemophilus influenza (Fleischmann, R. D., et al, (1995). Science 269:496-512), Phormidium laminosum (Packer, J. C., et al, (1995). Plant Mol. Biol. 27:199-204. K. Cregg, et al: Signal peptidase from Staphylococcus aureus Manuscript JB765-96), Bradyrhizobium japonicum (Mxc3xcller, P., et al, (1995). Mol. Microbiol. 18:831-840), Rhodobacter capsulatus (Klug, G., at al, (1996). GenBank entry, accession number 268305), Bacillus subtilis (two chromosomal and two of plasmid origin (Akagawa, et al, (1995) Microbiol. 141:3241-3245: Meljer, W. J. J. et al, (1995) Mol. Microbiol. 17:621-631; van Dijl, J. M., et al, (1992). EMBO J. 11:2819-2828), Bacillus licheniformis (Hoang, V., et al, (1993). Sequence P4266submitted to emb1/genbank/ddbj data banks.), Bacillus caldotyricus (van Dijl. J. M. (1993). Sequence p41027, submitted to emb1/Genbank/ddbj data banks). Bacillus amyloliquifaciens (two chromosomal genes) (Hoang, V. and J. Hofemeister, (1995). Biochim. Biophys. Acta1269:64-68; van Dijl. J. M. (1993). Sequence p41026, submitted to emb1/genbank/ddbj data banks) and a partial sequence has been reported for Bacillus pumilis (Hoang, V. and J. Hofemeister, (1995). Biochim. Biophys. Acta1269:64-68). These enzymes have been collectively defined as type-1 signal pepidases (van Dijl. J. M., et al, (1992). EMBO J. 11:2819-2828). Although the amino acid sequences of fifteen bacterial SPases (and a sixteenth partial sequence) have now been reported, the best studied examples are leader peptidase (LPase or LepB) from E. coli and a SPase from B. subtilis (SipS).
It has been demonstrated that LPase activity is essential for cell growth in E. coli. Experiments whereby expression of the lepB gene, encoding LPase, was regulated either by a controllable ara promoter (Dalbey, R. E. and Wickner. 260:15925-1593 1) or by partial deletion of the natural promoter (Date, T. (1983). J. Bacteriol. 154:76-83) indicated that minimization of LPase production was associated with cessation of cell growth and division. In addition, an E. coli strain possessing a mutated lepB gene (E. coli IT41) has been shown to have a drastically reduced growth rate and display a rapid and pronounced accumulation of preproteins when the temperature of the growth medium is elevated to 42xc2x0 C. (Inada, T., et al, (1988). J. Bacteriol. 171:585-587). These results imply that there is no other gene product in E. coli that can substitute for LPase and that lepB is a single-copy gene in the E. coli chromosome. This is in contrast to at least two species within the G+ Bacillus genus, B. Subtilis and B. amyloliquifaciens. It is known that there are at least two homologous SPase genes in each of these Bacillus species. The sipS gene can be deleted from the chromosome of B. subtilis 168 without affecting cell growth rate or viability under laboratory conditions to yield a mutant strain that can still process prexcex1-amylase. A putative SPase sequence (Akagawa, et al, (1995) Microbiol. 141:3241-3245)4.) may be the gene-product responsible for this activity and/or B. subtilis may harbor more than two SPase genes. Two or more genes encoding distinct SPase homologues reside on the chromosome of the closely related species B. amyloliquifaciens (Hoang, V. and J. Hofemeister, (1995). Biochim. Biophys. Acta 1269:64-68) and there is evidence to suggest that B. Japonicum may possess more than one SPase (Mxc3xcller, P., et al, (1995). Mol. Microbiol. 18:831-840; Mxc3xcller, P., et. al, (1995). Planta 197:163-175). Although SPase sequences from seven general of G+ bacteria are now known, only the single Bacillus genus amongst the G+ bacteria has been investigated with respect to SPase characteristics. It was therefore considered of interest to determine whether a G+ eubacterium that, unlike B. subtilis and B amyloliquifaciens, is not known for exceptional secretion activity has genes encoding more than one SPase with overlapping substrate specificities or whether it resembles E. coli and H. influenzae (and possibly other Gxe2x88x92 eubacteria)more closely in that it has a single SPase gene. The recent publication of the entire genomic sequence of the obligate G+-like intracellular bacterium Mycoplasma genitalium also reveals an interesting feature relating to heterogeneity amongst SPases (Fraser, C. M., et al, (1995). Science 270:397-403). Inhibitors of E. coli LPase have been reported (Allsop, A. E., et al. 1995. Bioorg, and Med. Chem. Letts. 5:443-448).
Evidence has accumulated to suggest that LPase belongs to a new class of serine protease that does not utilize a histamine as a catalytic base (Black., M. T., et al, (1992.). Biochem. J. 282:539-543; Sung, M. and R. E. Dalbey, (1992). J. Biol. Chem 267:13154-13159) but may instead employ a lysine side-chain to fulfill this role (Black, M. T. (1993). J. Bacteriol. 175:4957-4961; Tschantz, W. R., et al, (1993). J. Biol. Chem. 268:27349-27354). These observations and comparisons with Lex A from E. coli led to the proposal that a serine-lysine catalytic dyad, similar to that thought to operate during peptide bond hydrolysis catalyzed by LexA (Slilaty, S. N. and J. Little, (1987). Proc. Natl. Acad. Sci. USA 84:3987-3991), may operate in LPase (Black, M. T. (1993). J. Bacteriol. 175:4957-4961). Similar observations have since been made for SPase from B. subtilis (van Dijl, J. M., et al, (1995). J. Biol. Chem. 270:3611-3618) and for the Tsp periplasmic protease from E. coli (Keiler, K. C. and R. T. Sauer. (1995). Biol. Chem. 270:28864-28868); the similarities of SipS to LexA have been suggested to extend to several regions of primary structure (van Dijl, J. M., et al, (1995). J. Biol. Chem. 270:3611-3618). The serine and lysine residues (90 and 145 respectively in E. coli LPase numbering) known to be critical for catalytic activity in both E. coli LPase (Black. M. T. (1993). J. Bacteriol. 175:4957-4961; Tschantz, W. R., et al, (1993) J. Biol. Chem. 268:27349-27354) and B. subtilis SPase (van Dijl, J. M., et al, (1995). J. Biol. Chem. 270:3611-3618) and thought to form a catalytic dyad are both conserved in the S. aureus protein SpsB (S36 and K77). In addition, the aspartate at position 155 (280 in E. coli LPase numbering) is also conserved (this residue appears important for activity of the SipS SPase (van Dijl, J. M., et al. (1995.). J. Biol. Chem. 270:3611-3618) but less so for LPase from E. coli (Sung, M. and R. E. Dalbey, (1992). J. Biol. Chem 267:13154-3159). The present invention provides a novel SPase from S. aureus. 
Clearly, there is a need for factors that may be used to screen compounds for antibiotic activity and which may also be used to determine their roles in pathogenesis of infection, dysfunction and disease. There is a need, therefore, for identification and characterization of such factors which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases,
The polypeptide of the present invention has amino acid sequence homology to known serine proteases.
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel spsA by homology between the amino acid sequence set out in FIG. 2 and known amino acid sequences of other proteins such as Bacillus subtillis sipS.
It is a further object of the invention, moreover, to provide polynucleotides that encode spsA, particularly polynucleotides that encode the polypeptide herein designated spsA.
In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises the region encoding spsA in the sequence set out in FIG. 1 [SEQ ID NO:1], or a fragment, analogue or derivative thereof.
In another particularly preferred embodiment of the present invention there is a novel serine protease protein from Staphylococcus aureus comprising the amino acid sequence of FIG. 2 [SEQ ID NO:2], or a fragment, analogue or derivative thereof.
In accordance with this aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus bacterial clone contained in NCIMB Deposit No. 40771.
In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding spsA, particularly Staphylococcus spsA, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives, and compositions comprising same.
In accordance with another aspect of the present invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of spsA and polypeptides encoded thereby.
In accordance with this aspect of the invention there are provided novel polypeptides of Staphylococcus referred to herein as spsA as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing, and compositions comprising same.
Among the particularly preferred embodiments of this aspect of the invention are variants of spsA polypeptide encoded by naturally occurring alleles of the spsA gene.
In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned spsA polypeptides.
In accordance with yet another aspect of the present invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia: assessing spsA expression: to treat upper respiratory tract (e.g. otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g. empyema, lung abscess), cardiac (e.g. infective endocarditis), gastrointestinal (e.g. secretory diarrhoea, splenic abscess, retroperitoneal abscess). CNS (e.g. cerebral abscess), eye (e.g. blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g. epididymitis, inuarenal and perinephric abscess, toxic shock syndrome), skin (e.g, impetigo, folliculitis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis) bone and joint (e.g. septic arthritis, osteomyelitis); assaying genetic variation; and administering a spsA polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a Staphylococcus.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybridize to spsA polynucleotide sequences.
In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against spsA polypeptides.
In accordance with vet another aspect of the present invention, there are provided spsA antagonists which are also preferably bacteriostatic or bacteriocidal.
In a further aspect of the invention there are provided compositions comprising a spsA polynucleotide or a spsA polypeptide for administration to a cell or to a multicellular organism.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.